Micro-RNA (miRNA) is an endogenously expressed small (approximately 20- to 24-nucleotide) regulatory noncoding RNA, and regulates expression of various target genes at the post-transcriptional level as a component of RNA-induced silencing complex (RISC). When a miRNA is completely complementary to its target sequence in an mRNA, the miRNA induces cleavage of the mRNA, and causes rapid decrease in the mRNA level. However, most mammalian miRNAs have only a limited level of complementarity with target sequences located in the 3′-untranslated region (3′-UTR), and cause either translational repression or rapid deadenylation of target mRNAs in cytoplasmic processing bodies (P bodies). One-third or more of human coding genes are predicted to be targets of miRNAs (Bartel, D. P. (2004) Cell, 116, 281-297; Lewis, B. P. et al. (2005) Cell, 120, 15-20; Ambros, V. et al. (2003) Rna, 9, 277-279), and there is continuing evidence that shows miRNAs play important roles in differentiation, development, tumorigenesis, and cellular defense against infection (Li, Q. J. et al. (2007) Cell, 129, 147-161; Lu, J. et al. (2005) Nature, 435, 834-838; Lecellier, C. H. et al. (2005) Science, 308, 557-560).
Techniques to specifically inhibit the activity of miRNA molecules are considered indispensable for their global functional analysis. Several methods for inhibiting miRNA function already exist, and for example, chemically modified single-stranded oligonucleotides such as 2′-O-methyl (2′-OMe) RNA (Hutvagner, G et al. (2004) PLoS Biol, 2, E98; Meister, G et al. (2004) Rna, 10, 544-550), locked nucleic acid (LNA), and “antagomirs” (Orom, U. A. et al. (2006) Gene, 372, 137-141; Krutzfeldt, J. et al. (2005) Nature, 438, 685-689) are used. These reagents are chemically synthesized to have complementarity towards mature miRNAs. They are resistant to cellular nucleases, and may function as substrates not cleaved by RISC. Since they are designed to be introduced into cells by transfection, the inhibitory activity is inevitably transient.
Recently, DNA vectors that express “microRNA sponges” which are competitive inhibitors of miRNAs were reported (Ebert, M. S. et al. (2007) Nat Methods, 4, 721-726). Although transient expression of microRNA sponge vectors efficiently inhibits miRNA function, the inhibitory effects are not maintained for more than one month even if transfection is carried out using DNA-based vectors. Therefore, it is desirable to establish methods for inhibiting miRNAs for a longer period of time.